Construction Of The Head Transcriptome Database And Chemosensory Gene Mining And Analysis Of The Head Locust

Chemosensory genes play an important role in foraging,finding mates,locating hosts,selecting oviposition sites,and escaping from natural enemies.The process of detection and identification of insects to the extermal scent molecules is mainly related to several proteins,such as odorant binding proteins,chemosensory proteins,odorant receptor proteins and gustatory receptor proteins.These proteins can be used as new targets to interfere the recognition of odors and affect insects' normal activities during the insect pest control procedure.The bamboo locust,Ceracris kiangsu(Orthoptera:Arcypteridae)is a serious insect pest of bamboo in China,which harm the bamboo resouces in southern China and hinder the development of forestry.Therefore,in-depth study of molecular mechanism of C.Kiangsu chemosensory genes will lay an important foundation for the specific pest control.However,during the recent years,chemical control has been mainly undertaken to prevent the bamboo locust,but the bamboo locust could beome resistant to insecticides.So searching for new biological control technology has become current research hotspot.In this study,to obtain more data for identifying and analyzing the chemosensory genes,high-throghput sequencing was firstly used to analyze the head transcriptome of C.kiangsu.The results lay the foundation for the future research of the gene function,and provides theoretical support for the biological control of C.Kiangsu.The main results and conclusions are as follow:1.Transcriptome database of the heads of C.Kiangsu.Using the Illumina sequencing,we obtained more than 138,381,768 sequencing reads derived from the heads of C.kiangsu.After data filtering,133,790,136 clean reads were yielded.These reads were assembled into 260,822 unique sequences(mean size=814 bp).Of the unigenes,90,923(34.86%)had significant similarity with proteins in the NCBI nonredundant protein database and Swiss-Prot database(E-value<10-5).Of these annotated unigenes,82,462 were assigned to gene ontology categories.Of them,assignments to the biological process made up the majority(39,572,47.98%),followed by molecular function(24,067,22.82%)and cellular component(18,823,28.80%).In total,13,354 unigenes were mapped onto 281 pathways using the Kyoto Encyclopedia of Genes and Genomes Pathway database(KEGG).In addition,our transcriptional analysis identified 46,609 short sequences as potential SSR markers,for SNP,a total of 163,427 polymorphic loci were found.2.The discovery and analysis of the chemosensory genes.From the data base of transciptome,we detected 8 putative odorant binding protein(OBPs),3 pupative chemosensory protein(CSPs),3 putative odorant receptor genes(ORs),and 1 putative gustatory receptor(GR)of C.kiangsu for the first time.Phylogenetic trees were generated to show the relationships among the chemosensory genes of C.kiangsu and other incect species.Our results showed that the relationships of chemosensory genes between Orthoptera and Hemiptera are most closest,which in accordance with the evolution analysis of the insects.The relative expression levels of OBPs were determined by qRT-PCR with different tissues(head,antenna,mouthpart,body and leg).The results indicated that all of these genes were highly expressed in antennae.And we found that CkiaOBP2 was highly expressed in all five tissues,which indicates that this gene may play an important role in variety of physiological activities.3.Total length amplification and analysis of olfactory co-receptor gene.The olfactory receptor co-receptor gene of C.kiangsu(CkiaOrco)was cloned for the first time with the technology of RACE.The open reading frame of CkiaOrco is 1,458 bp in length,encoding 485 amino acid.And we analysised its physical and chemical properties.Phylogenetic analysis showed that species of each order had a separate branch.According to the relationship of differentiation,the order is Hemiptera,Orthoptera,Hymenoptera,Lepidoptera,Coleoptera and Diptera.And in the branch of Orthoptera,compared with the Locusta migratoria,the relationship of C.kiangsu and Schistocerca gregaria is more close.The result of adaptive evolution showed that Oreo genes of insects were general under negative selection.And we found three sites of mature protein region(230EA,243D and 307E)were under positive selection.In addition,we analysised the gene expression profiling of CkiaOrco,the data indicated that the gene expressed in all tissues,and had no remarkable difference between male and female.